Project Summary/Abstract Ocular pharmaceuticals predominantly use the topical route of administration, which involves a number of benefits and limitations when compared to systemic drug routes. A major limitation of the ocular topical route is the rapid loss of drug via tearing and nasolacrimal drainage, requiring the use of frequent dosing and high drug concentrations that then create formulation difficulties, together with the potential for local and systemic adverse effects. The proposed innovation is to use the temperature sensitivity of the genetically engineered smart polymer class of elastin-like polypeptides (ELP) to control ocular clearance. ELPs have unique properties that promote phase separation, recombinant expression, protein purification, and self-assembly of nanostructures. ELPs are repeated pentameric peptides, (VPGXG)n, that have characteristic inverse phase transition temperatures, Tt, above which they phase separate from aqueous solution. By selecting X and the length n, ELPs of different Tt can be efficiently and precisely biosynthesized. Genetically engineered ELPs are pharmacologically relevant, being monodisperse, biodegradable, and biocompatible. Lacritin, a new-found, short glycoprotein promotes tear secretion in dry eye models but must be administered several times a day. Lacritin will be genetically fused with ELPs of different transition temperatures and molecular weights to optimize the construct for drug retention. Free, active lacritin is expected to be in equilibrium with the ELP aggregates, thus extending ocular lacritin residency. ELPs with a transition temperature greater than 37C should clear quickly from the eye; however, ELPs with transition temperature between room temperature (25C) and body temperature (37C) are expected to drain slowly. The following specific aims are designed to serve as a proof of concept of this idea: SA1) Synthesis and in vitro demonstration of lacritin-ELP thermal sensitivity. Thermally sensitive and insensitive ELPs with and without fluorescent labels will be prepared with molecular weights ranging from 10 to 50 kD and fused to lacritin. Our milestone will be to produce a thermally sensitive ELP-lacritin that will have a transition temperature of between 25 and 37C, suitable for use in an eye drop. SA2) Efficacy and tolerability evaluation of lacritin-ELP in in vivo normal rabbit and in vitro Ussing chamber ocular models. Optimal fusion peptides will be evaluated for residency, ocular tolerability and lacritin-stimulated tear formation in in vivo normal rabbit models and in in vitro Ussing chamber residency experiments. Our milestone is to demonstrate that the thermally-sensitive lacritin-ELP is more effective than the thermally-insensitive product at increasing tear production without significant local or systemic toxicity.